Dr. Srour proposes to examine the relationship between cell cycle progression and cell division of hematopoietic stem cells (HSC) and maintenance of fundamental function of these cells. During the previous funding period, he gained insight into the association between mitotic quiescence and HSC function and correlated several biological properties of these cells with their position in the cell cycle. He documented a decline in hematopoietic potential as dormant CD34+ cells traversed from G0 into G1 in the absence of cell division and illustrated the existence of a hierarchical organization of HSC relative to their responsiveness to cytokine stimulation. Strategies delivering efficient retroviral mediated gene transduction and long-term expression of transduced genes were formulated. Although Dr. Srour gained valuable information by meeting or exceeding all of the specific aims of the original proposal, more remains to be learned. The relevance of mitotic quiescence of HSC to their functional potential during embryonic development is yet to be explored. Dr. Srour contends that it remains to be established whether HSC fate is intrinsically or extrinsically regulated. To better understand these critical issues in stem cell biology, three specific aims will be examined. First, Dr. Srour will test whether during embryonic development, non-cycling, as well as HSC in active phases of the cell cycle, are equally effective in engrafting and sustaining long-term hematopoiesis. He hypothesizes that the extensive need for hematopoietic cells during fetal development obligates resting and cycling HSC to contribute to blood cell production while maintaining the stem cell pool. Second, he will investigate if recruitment of dormant HSC into active phases of the cell cycle and subsequent self-renewal divisions are intrinsically or extrinsically regulated. Third, he will use information gleaned from the first and second specific aims to improve the transduction efficiency of foreign genetic material into HSC using retroviral mediated gene transfer. These specific aims should yield valuable information about normal hematopoiesis and the mechanisms that regulate stem cell fate. Information gained from these studies will be important for understanding the roles of cell cycle activation and proliferation in maintaining the hematopoietic potential of HSC throughout ontogeny, and for proper utilization of HSC in transplantation, ex vivo expansion, and gene therapy.